Photoaffinity labeling of the two forms of serotonin binding protein: peptide mapping of the binding sites.
Serotonin binding protein (SBP) is a vesicular protein found in neurectoderm-derived cells that store 5-hydroxytryptamine (5-HT, serotonin), such as central and peripheral serotonergic neurons and paraneurons (parafollicular cells of the thyroid). 5-HT is stored as a complex with SBP in vivo. Two forms of the protein are found. These differ in molecular mass: one is 45 kDa and the other 56 kDa. It has been suggested that the 56-kDa form of SBP may be the precursor of the 45-kDa form. To study the relationship between these two proteins, we have used a covalently bound radiolabeled probe to analyze their binding domains. A photoaffinity reagent, N-(4-azido-2-nitrophenyl)-5-hydroxytryptamine (NAP-5-HT), was synthesized and characterized by nuclear magnetic resonance spectroscopy, mass spectra, and UV-visible absorption spectra. A 1 M excess of NAP-5-HT inhibited the binding of [3H]5-HT to SBP by 50%. NAP[3H]5-HT was also synthesized and attached to both high- and low-affinity binding sites on both forms of SBP. The high-affinity constants for 45-kDa and 56-kDa proteins were 0.8 nM and 0.02 nM, respectively, whereas the low-affinity constants were 0.3 microM and 0.15 microM. When the high-affinity site of partially purified SBP was photoaffinity-labeled with the reagent, two covalently labeled proteins (45 kDa and 56 kDa) were found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of the labeling of both proteins by 50% was observed in the presence of a 15-fold molar excess of 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)
Liu
K P
KP
Division of Neuroscience, New York State Psychiatric Institute, NY 10032.
Tamir
H
H
Adlersberg
M
M
D000345:Affinity Labels; D001386:Azides / Q000138:chemical synthesis; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D019286:Endopeptidase K; D008027:Light; D008666:Metalloendopeptidases / Q000378:metabolism; D010449:Peptide Mapping; D012697:Serine Endopeptidases / Q000378:metabolism; D012701:Serotonin / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; C025366:serotonin-binding protein; D012701:Serotonin; C063111:N-(4-azido-2-nitrophenyl)-5-hydroxytryptamine; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus